1) MtDNA Haplogroup H has been found in Paleolithic remains, which is a big support for continuity of Paleolithic populations in Europe.

2) haplogroup N1a was absent in the Neolithic groups analysed here, which might suggest that the genetic influence of Neolithic groups from the Near East in Central Europe was different to that in the western Mediterranean area (LBK vs Cardial?)

3) In sum, the differences between Hunter-Gatherer and Neolithic groups in Europe can be attributed to the restructuring of their genetic make-up due to the incoming gene flow from the Near East. The genetic data obtained in this mtDNA study contradicts the total acculturation and replacement models proposed for explaining the phenomenon of Neolithisation. Whereas in Central Europe-Northern France a Neolithic clinal gradient can not be discarded from aDNA data so far, the differences in the mtDNA found between Neolithic sites at the Mediterranean area provide support for a random dispersion model. Maritime colonization, transporting small and different Neolithic groups from the Near East pool could contribute to explain the difference.[/b]

1) MtDNA Haplogroup H has been found in Paleolithic remains, which is a big support for continuity of Paleolithic populations in Europe.

I have published many posts, here and elsewhere, also against the last paper of Behar et al. about hgs. H, but also R0a, HV, K etc.There are 3 hg. H which are probably the witnesses of the most ancient H, like some outliers I have found for some Y: 1 is from France, the second belong to an American woman adopted, whose autosomal is at least 60% Italian. I hope to ascertain soon her maternal origin. Anyway probably nothing from Middle East.

This is from Shlush, then from Middle East, but as I think having demonstrated about the H26 I have spoken about on another thread, it may have come from EuropeSample Id: EU600353 Unresolved Options: H15b;H56a;Partial Descendants: Private Mutations: G54c; T55a; A200G; T3847C; T6253C; A9336G; G9612A; A9644G; A10819G; G13145A; T16086C; Topologically Missing: Country: Geography: Ancestry: Reference: Shlush 2008 Contact:

1) MtDNA Haplogroup H has been found in Paleolithic remains, which is a big support for continuity of Paleolithic populations in Europe.

Unfortunately this is just the usual problem of reporting CRS as H. Really it should be recognised by now, but here we have the exact same issue. Once researchers switch over to the new system for mtDNA that does not involve CRS, results will become more reliable.

Of the three Magdalenian results, one was rCRS, naively reported as H, but could be a number of things, one was 16093, 16362, reported as H6, but more probably R0a1b at that date and the other was U5.

The one Mesolithic result was U5b1, in line with findings in Mesolithic remains elsewhere in Europe.

Of the Neolithic remains, 8 were rCRS reported as H, others reported H on the basis of just one mutation.

It is really very irritating that there was almost no testing beyond HVR-I: 15,998–16,400, and HVR-II,16504-429.

Hey! That T2 from Navarre could be a relation of mine! My line is T2g and traces to Brittany in 1698. My Breton relation has the name "Arguin", which means "stone cutter" in Basque. Maybe there's a Basque/Breton connection.

Unfortunately this is just the usual problem of reporting CRS as H. Really it should be recognised by now, but here we have the exact same issue. Once researchers switch over to the new system for mtDNA that does not involve CRS, results will become more reliable.

Of the three Magdalenian results, one was rCRS, naively reported as H, but could be a number of things, one was 16093, 16362, reported as H6, but more probably R0a1b at that date and the other was U5.

Not quite, if you look at their table-S1, you will see that some rCRS was reported as U, whereas other was reported as H. This wasn't done naively, the rCRS were assigned as H when they tested positive for base pair A instead of G in the position 73 of the HVR-II, and also when they tested negative for U RFLPs analyses. So no, this isn't one of those papers where rCRS is just assumed to be H for the heck of it. Check Table-S1 and you will notice how some rCRS are U, and some are H.

For example there is HVR-I rCRS which has HVR-II position 73 base pair A, and is listed as H and haplotype 2. There is HVR-I rCRS whic has HVR-II position 73 base pair G, and HVR-II 73-146-263-285-309.1-310.1-312-313 and is also listed as H and haplotype 3. Finally there is There is HVR-I rCRS whic has HVR-II position 73 base pair G, and HVR-II 73-146-263-309.1-315.1-320 and is actually listed as U and haplotype 4.

As for H6, well it was tested using the HVR-I and RFLPs analyses, so it likely yielded negative results for R0 or HV.

The one Mesolithic result was U5b1, in line with findings in Mesolithic remains elsewhere in Europe.

Of the Neolithic remains, 8 were rCRS reported as H, others reported H on the basis of just one mutation.

It is really very irritating that there was almost no testing beyond HVR-I: 15,998–16,400, and HVR-II,16504-429.

Well, they did test for RFLP and the HVR-II position 73.

Quote from: Hervella et al(2012)

Sequencing of mtDNA HVR-I, nucleotide positions (nps) 15,998–16,400, and mtDNA HVR-II, nps 16504-429 as per [61], was undertaken in six overlapping fragments, each with a length of approximately 100 bp (base pair). HVR-II sequencing was carried out in samples with no polymorphic positions in HVR-I (Table S6). Similarly, the fragment between primers 8F and 8R (Table S6) was amplified in all samples to determine position 73 of HVR-II. The PCRs were performed in 25 μl of reaction mixture containing 10 mM Tris-HCl pH 8.3; 2 mM of MgCl2, 0.1 μM of each dNTP, 0.4 μM of each primer, 5 units of AmpliTaq Gold (Applied Biosystems) and 10 μl of diluted DNA (1 μl of DNA extract in 10 μl of 1 mg/ml BSA). Cycling parameters were 95°C for 10 min; followed by 40 cycles of 95°C for 10 sec, annealing temperature for 30 sec, 72°C for 30 sec; and a final step of 72°C for 10 min. The annealing temperatures of the primers of HVR-I were as follows: 60°C for the A1/A1R primer pair, 58°C for 2F/2R and 4F/4R, 57°C for 1F/1R and 55°C for 3F/3R and 5F/5R, the primer sequences are listed in [62]. The sequence and annealing temperatures of the HVR-II primers are shown in Table S6. The amplification of each fragment was undertaken in independent PCRs and each fragment was amplified and sequenced twice from two independent DNA extract. In the case of positive amplification and the absence of contamination, the amplifications were purified by ExoSAP-IT (USB Corporation), with subsequent sequencing in an ABI310 automatic sequencer using chemistry based on dRhodamine. The results obtained were edited with BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit​.html) and the sequences were aligned manually.

In order to classify the mitochondrial variability of the individuals analyzed in this study, we proceeded to amplify 11 markers, which are required for defining the 10 Caucasian haplogroups described [63]. The protocol and primers are described in [17], [20], [42]. The digestion patterns were verified using a fragment Bioanalyzer (Agilent Technologies).

Not quite, if you look at their table-S1, you will see that some rCRS was reported as U, whereas other was reported as H. This wasn't done naively, the rCRS were assigned as H when they tested positive for base pair A instead of G in the position 73 of the HVR-II, and also when they tested negative for U RFLPs analyses. So no, this isn't one of those papers where rCRS is just assumed to be H for the heck of it. Check Table-S1 and you will notice how some rCRS are U, and some are H.

For example there is HVR-I rCRS which has HVR-II position 73 base pair A, and is listed as H and haplotype 2. There is HVR-I rCRS whic has HVR-II position 73 base pair G, and HVR-II 73-146-263-285-309.1-310.1-312-313 and is also listed as H and haplotype 3. Finally there is There is HVR-I rCRS whic has HVR-II position 73 base pair G, and HVR-II 73-146-263-309.1-315.1-320 and is actually listed as U and haplotype 4.

Jean M is right, if the haplotype is rCRS for HVR-I and Not Analysed for HVR-II, the sample is assumed to be H. But we need the HVR-II and the coding region results to know the real haplogroup.

Jean M is right, if the haplotype is rCRS for HVR-I and Not Analysed for HVR-II, the sample is assumed to be H. But we need the HVR-II and the coding region results to know the real haplogroup.

They did test the HVR-II and the coding region, what part of the n73 column on Table-S1 is so hard to understand. When a haplogroup is rCRS in the HVR-I, one can test the position 73 on the HVR-II to see if it has an "A" thus indicating that it is definitely H, or a "G" which could mean that it could be either H or U. Then in order to tell them apart, one could test more positions on the HVR-II, and also test the FRLPs to determine if is H or U. All of these tests were performed; the coding region was tested for FRLP in all haplogroups. Did you guys even bothered reading what I posted above, where the authors clearly say that the amplified 11 markers that are necessary to indentify 10 Caucasian haplogroups, and provide references on how they did it. I don’t know if Jean M is doing it purposely or if she missed that part, but what is clear is that this wasn’t one of those “if is rCRS it must be H” studies, they did three different tests to correctly assign some of rCRS to H and some to U.

For example, if your HVR-1 result shows that you have no mutations compared to CRS, the possible haplogroups that you may belong to are H, HV, Pre-HV, R, and U. If your HVR-2 results show a mutation at position 73, then the ranking for Haplogroup R and U will become higher since Haplogroup H, HV and Pre-HV do not contain a mutation at position 73. If you want to confirm whether you belong to Haplogroup R or Haplgroup U, the Haplogroup Backbone SNP Test, which examines markers in the coding region of the mtDNA, will uncover SNP marker 12308 and allows you to confirm whether you are a member of Haplogroup U or R in this example.

So because the HVR-II did not have mutation(A=>G) at position 73, this means that it highly unlikely to be R or U, then a way to determine if is R0, HV or U is to test for Alu I 7025, if tests negative for Alu I 7025 then it is definitely H and not R0 or HV. That is exactly what they did.

I don’t know if Jean M is doing it purposely or if she missed that part, but what is clear is that this wasn’t one of those “if is rCRS it must be H” studies, they did three different tests to correctly assign some of rCRS to H and some to U.

In Italian we say that "Non c'è peggior cieco di chi non vuol vedere". They (Jean Manco, but also Dienekes and infinite others) don't see "pour cause": all their work is becoming nothing. I would say what I said to Anatole Klyosov: an impressive waste of time!Imagine when some aDNA R1b (and why not R1a) will spring up in some place of Western Europe!

OK I am looking at table S1.You will notice that the top result CAS-1 has rCRS in HVRI, then G for 73 and did not test HVRII. It was reported as H. In fact they only tested the HVRII for 11 samples.

What am I missing? If they did anything else, where is the data?

[Added] I see that they got 73A for PS1-1, which should mean that is H or V or HV or at least on the RO tree. Will correct.

You are missing this statement which is found in the subsection called: "Analysis of the variability of mtDNA"

Quote from: Hervella et al.2012

In order to classify the mitochondrial variability of the individuals analyzed in this study, we proceeded to amplify 11 markers, which are required for defining the 10 Caucasian haplogroups described [63]. The protocol and primers are described in [17], [20], [42]. The digestion patterns were verified using a fragment Bioanalyzer (Agilent Technologies).

If you look at the references you will see that one of them [17], is a study done by several of the authors on this study. The study is called:

In order to classify the variability of the mitochondrial genome of the individuals buried in Aldaieta we proceeded to amplify and type 11 markers, which are required for defining the 10 haplogroups that have been described in Caucasoids (Macaulay et al. 1999).

The amplification involved the design of a pair of primers for each marker that amplify a product of 100–120 bp in length, approximately. Table 2 shows the list of primers, the annealing temperature of each one and the size of the amplified product.

Those are the 11 markers that were used to perform RFLPs analyses on the coding region. That is the missing key information which tell us how they were able to determine that for example CAS-21 even though it did have the mutation on HVR-II 73 probably tested negative for the RFLP which would make it U.

I agree that the authors should have been more explicit and perhaps provide a Table with the RFPL primers used, but it seems they referenced it to previous studies, and mentioned that they used those 11 markers.

So how can they be certain that PS1-1 is H and not HV, or R0 because it tested negative for Alu I 7025, thus making it indisputably H.

one can test the position 73 on the HVR-II to see if it has an "A" thus indicating that it is definitely H

A at position 73 is not indicating that it is definitely H. HVR-II means High Variable, so we find often back mutation at the position 73. I am sure that we can find different H sequences with different values at position 73. This is true for all the haplogroups.

... and also test the FRLPs to determine if is H or U. All of these tests were performed; the coding region was tested for FRLP in all haplogroups. Did you guys even bothered reading what I posted above, where the authors clearly say that the amplified 11 markers that are necessary to indentify 10 Caucasian haplogroups, and provide references on how they did it.

Sorry but for me it is not very clear when I read we proceeded to amplify 11 markers, which are required for defining the 10 Caucasian haplogroups described

If I don't have the data, I can't include it in my table. Frankly I'm not willing to take statements on trust. I can (and do) state the reported haplogroups in the column with supplied alleles, but my column on the haplogroup will reflect the haplogroups as best I can assign them on the data, or have a query.

Sorry but for me it is not very clear when I read we proceeded to amplify 11 markers, which are required for defining the 10 Caucasian haplogroups described

Maybe do your homework, and look at the references they provide when they say:

"In order to classify the mitochondrial variability of the individuals analyzed in this study, we proceeded to amplify 11 markers, which are required for defining the 10 Caucasian haplogroups described [63]. The protocol and primers are described in [17], [20], [42]. "

Those are the 11 markers that were used to perform RFLPs analyses on the coding region. That is the missing key information which tell us how they were able to determine that for example CAS-21 even though it did have the mutation on HVR-II 73 probably tested negative for the RFLP which would make it U.

I agree that the authors should have been more explicit and perhaps provide a Table with the RFPL primers used, but it seems they referenced it to previous studies, and mentioned that they used those 11 markers.

So how can they be certain that PS1-1 is H and not HV, or R0 because it tested negative for Alu I 7025, thus making it indisputably H.

Thanks for these informations, but H is defined by mutations G2706A and T7028C. What is Alu I 7025 ?

If I don't have the data, I can't include it in my table. Frankly I'm not willing to take statements on trust. I can (and do) state the reported haplogroups in the column with supplied alleles, but my column on the haplogroup will reflect the haplogroups as best I can assign them on the data, or have a query.

Well you are grasping for straws now, I'm sorry but if it clearly says on the paper they used 11 RFLPs markers, and they are referencing the protocol and primers in previous studies, then to say that they don't provide the data is just bogus. You are just basing off your skepticism on the fact to you buy into the whole Haplogroup H is Neolithic trend.

So what is more likely:

1-The authors did test for RFLPs as they mention in the study, and this is why CAS-21 even though it is rCRS, and has HVR-II position 73 G is listed as H, probably because it failed to provide the primers that would make it U.

2-They just felt like randomly assigning some of rCRS sequences with HVR-II position 73 G to U and the other to H just based on a hunch.

Maybe this will clarify how the rCRS HVR-II 73 G combo gets assigned to H instead of U.

We report on the use of rare mutations to tackle biosocial questions such as kinship and differential burial practices from past human populations. To do this, we have inferred nucleotide position 73 of HVS-II in individuals classified as belonging to haplogroup H from 76 human dental samples from the necropolis of Aldaieta (Basque Country, Spain, 6th-7th century) by means of PCR and restriction enzyme tests. The same analysis has been performed for 146 extant individuals from the northern Iberian peninsula. A combination of haplotype H and 73G in HVS-II, rare in extant populations (0.5-3%), has been found at a frequency of 20% in the ancient population of Aldaieta. These data can be explained in terms of the existence of different burial practices associated with a variety of factors, mainly social status and kinship. This hypothesis is also supported by archeological data. These results indicate that caution should be taken when making phylogenetic inferences from extinct populations, because an uncharacterized kinship can significantly bias allele frequencies.

Again if you two bothered to read the references they provided, instead of jumping on the bandwagon that there isn't enough information, you would have noticed that they did in fact test for RFPLs/restriction enzyme test.

Well you are grasping for straws now, I'm sorry but if it clearly says on the paper they used 11 RFLPs markers, and they are referencing the protocol and primers in previous studies, then to say that they don't provide the data is just bogus.

(Sigh). I'm simply stating a fact. If they have results from those markers, then they should provide them. That is standard practice. If they don't provide their results, then other researchers cannot see the reasoning behind their assignment to haplogroups. You can't just expect readers to take it on trust.

Thanks for these informations, but H is defined by mutations G2706A and T7028C. What is Alu I 7025 ?

Alu I 7025 is a RFLPs test, for example the sample from La Chora, Cantabria, which tested HVR-I 16093-16362, Jean Manco mentioned that it could also be R0a1b instead of the H6 that was reported. The way the authors could be able to differentiate between R0 and H6 would be do a restriction enzyme test, or RFLP analysis. If the sample turns out to be Alu I 7025 +, then it is definitely R0, because H is Alu I 7025-. You can look at Table-3 of this study to see how they can test for haplogroups using RFLPs.

Well you are grasping for straws now, I'm sorry but if it clearly says on the paper they used 11 RFLPs markers, and they are referencing the protocol and primers in previous studies, then to say that they don't provide the data is just bogus.

(Sigh). I'm simply stating a fact. If they have results from those markers, then they should provide them. That is standard practice. If they don't provide their results, then other researchers cannot see the reasoning behind their assignment to haplogroups. You can't just expect readers to take it on trust.

But they do, they are directing you to the studies where they used those 11 markers, and even give the primers. Is it too hard to click on the reference, and follow the instructions of the authors when they are telling you to refer to those studies for the protocol and primers of the 11 markers used?

I'm frankly baffled by the assignment of CH-1 from La Chora to H6, which should have G73A, T239C T16362C A16482G

Instead the sample has 73G, 16093 and 16362.

Tell you what, why don't you email them and ask them about it. I know Maju was concerned as to whether or not they had used RFPLs analyses, and he emailed them, and they already replied saying they did.

But they do, they are directing you to the studies where they used those 11 markers, and even give the primers. Is it too hard to click on the reference, and follow the instructions of the authors when they are telling you to refer to those studies for the protocol and primers of the 11 markers used?

The protocol and primers are not in question. That they have used them on other studies is absolutely fine. The fact that they do not provide the results of any such tests on the samples in this study is the issue.

Tell you what, why don't you email them and ask them about it. I know Maju was concerned as to whether or not they had used RFPLs analyses, and he emailed them, and they already replied saying they did.

Well maybe Maju can get the results out of them. I expect that he will pass them on to me if he does.